Studies at the behavioral level reveal that in both vertebrate and invertebrate species, memory consolidation involves processing of information through several distinct temporal "phases". At the anatomical level, memories appear in many cases to be dynamically transferred between distinct loci. Even elemental forms of associative learning in relatively simple invertebrate brains can rely on multiple mechanisms in relatively large networks of neurons. Central to this proposal is the availability in flies of a Gal4-responsive temperature-sensitive (and dominant negative) dynamin transgene (UAS-Shi/ ts), which permits temperature dependent and reversible disruption of neuronal function. This genetic tool provides a unique opportunity to functionally map the relationships between neural circuitry and a complex behavior such as memory. We already have used this method to disrupt neuronal function in several distinct sub-regions of the fly brain, thereby dissecting the anatomical requirements for acquisition versus retrieval as well as for storage of anesthesia-sensitive versus anesthesia-resistant memory. The goal of this proposal is to further define the functional circuitry underlying memory formation, storage and retrieval. The three Specific Aims of this proposal are: 1- "Functional anatomy"- Each of these 11 non-mushroom body Gal4 enhancer lines, as well as the DPM and MB lines, will be used to drive spatially restricted expression of the Gal4-responsive UAS-Shits transgene. Temperature-shift experiments then will be used to transiently disrupt neuronal function in these foci during each temporal phase of memory consolidation. 2- "Anatomical Dissection of Memory Phases" - Functionally relevant Gal4 lines identified in Aim #1 will be combined with the "phase specific" amnesiac and radish mutants. This "double mutant" approach will permit dissection of the anatomical requirements for amnesiac-dependent memory, which is anesthesia sensitive, from radish-dependant (anesthesia resistant) memory. 3- High Resolution Imaging- Confocal imaging will be used to construct a high-resolution anatomical map of GFP reporter expression for each Gal4 transposon line identified as functionally relevant in Specific Aims I and 2. We will take advantage of the economy-of-scale and molecular genetic tools available only in flies to investigate the relationships between temporal and spatial patterns of neural activity and the ongoing consolidation of memories in the brain. While the anatomical wiring diagram of nervous systems appear species specific, the molecular mechanisms and behavioral properties underlying memory within these circuits are remarkably conserved. As a result, an understanding of the "logic" of how information is processed in relatively simple nervous systems can greatly inform future investigations of human cognition.